In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. Structural defects of the heterotrimeric type I collagen molecule are well known to cause the dominant bone disorder osteogenesis imperfecta. A severe recessive form of OI was first postulated in 1979. More recently, investigators have noted that some patients with clinical OI do not have defects detected in the type I collagen genes during sequencing. These patients without mutations in collagen can be divided into those who have abnormal collagen biochemistry and those with normal electrophoretic migration of the collagen chains. We hypothesized that the cause of recessive OI with abnormal collagen biochemistry and normal collagen gene sequence would involve a gene(s) whose products interacted with type I collagen. Several years ago the BEMB identified defects in two components of the collagen prolyl 3-hydroxylation complex, CRTAP and P3H1 (encoded by LEPRE1) as the cause of recessive OI. Our work has generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects in the components of a complex in the endoplasmic reticulum that modifies collagen cause recessive OI. In the expanded nosology for OI, defects in CRTAP and LEPRE1 are designated as types VII (OMIM #610682) and VIII (OMIM #610915) OI, respectively. Recessive OI is now a major area of investigation for the BEMB. The phenotypes of types VII and VIII OI are distinct from classical dominant OI, but difficult to distinguish from each other. Both groups of children have severe/lethal OI with white sclerae, normal or small head circumference, rhizomelia, metacarpal shortening and severe undertubulation of long bones. Biochemically, both groups have normal collagen sequences with absence of 3-hydroxylation of the Pro986 residue, but full overmodification of the helical prolines and lysines by prolyl 4-hydroxylase and lysly hydroxylase. This overmodification of the helix was unexpected and indicates that absence of the components of the 3-hydroxylation complex leads to delayed folding of the collagen helix. We have now shown that the basis of the phenotypic and collagen biochemical similarity of types VII and VIII OI is that CRTAP and P3H1 are mutually protectively in the complex. We observed that CRTAP is severely reduced and P3H1 is absent from cell lysates which have a defect in either component of the complex, although the transcript level of the normal component is not reduced. The interpretation of mutual protection of CRTAP and P3H1 is supported by immunofluorescence microscopy;reduced levels of both proteins are detected in cells with a mutation in either gene. Mutual protection of components is also supported by the results of stable transfection of CRTAP expression constructs into transformed CRTAP-null cells. Expression of CRTAP restores P3H1, as well as CRTAP protein in these cells. Furthermore, there is a partial rescue of collagen modification in the stably transfected cells, which indicates that the components are functioning to restore collagen folding. P3H1 is partially rescued in CRTAP-null cells treated with inhibitors. Also, in LEPRE1-null cells, the secretion of CRTAP into conditioned media is increased compared with control cells and accounts for 15-20% of the decreased CRTP detected in cells. This year the BEMB identified a two children with a mutation in the 3rd component of the collagen 3-hydroxylation complex, CyPB which is incoded by PPIB. These siblings have recessivvee OI of moderate severity with white sclerae but without rhizomelia. They have a homozygous mutation in the start of codon of the peptidly prolyl isomerase gene, which results in a total absence of CyPB protein as measured on Western blots with 3 different antibodies and by immunfluoresence microscopy. Surprisingly, the 3-hydroxylation of collagen Pro986 and the hydroxylation of helical lysine and proline residues were both normal. First of all, this means that the two component of the 3-hydroxylation complex, CRTAP and P3H1, can complete collagen modification in the absence of the 3rd component. Second, normal helical modification indicates that the folding rate of the collagen helix is normal. Since CyPB had been previously thought to be the unique collagen cis-trans prolyl isomerase, normal collagen folding in the absence of CyPB means that there must be redundancy for this important function in human cells. Among our LEPRE1-deficient patients, the BEMB identified a common mutant allele, IVS5+1G to T, which occurred in both African-Americans and West African families which had emigrated to USA. This so-called "West-African allele" accounts for a third of the known LEPRE1 mutations, and has been found only in individuals of African descent. We hypothesized that this mutation had been transported to the Americas via the Atlantic slave trade. We obtained samples from 3 African American cohorts and determined a carrier frequency in Mid Atlantic USA of 1 in 200-300 African-Americans, typical for a rare recessive disorder. In a collaboration which Charles Rotimi of NHGRI we examined over 1200 DNA samples from contemporary Ghanians and Nigerians. To our surprise, this groups had a carrier frequency for this lethal recessive mutations of 1.5%! This high carrier frequency makes the inheritance of severe OI in African distinct from the dominant form prevalent in North America and Europe, where recessive OI occurs in 5-7% of OI cases. In West Africa, recessive is expected to account for about half of all severe OI. To determine the age of the mutation, we have conduction haplotype analysis on African North American pedigrees, yielding a 4.2 MB conserved region surrounding the LEPRE1 gene. The mutation is calculated to be about 600 years old, consistent with a founder mutation in West African that originated before the Atlantic slave trade. We are now conducting studies to determine geographic distribution of the mutation, whether it is limited to Ghana/Nigeria or is pan-African.